1. Field of the Invention
This invention relates to a method for reducing the carbon dioxide background due to blood metabolism in a bacterial medium by the addition of a mixture of saponin and a phospholipid to maintain the lytic activity of saponin over a broad concentration range of blood cholesterol levels in the sample and preserve the lytic activity of saponin containing media after heat sterilization.
2. Background of the Related Art
Automated diagnostic instruments such as the Bactec.RTM. 460, the Bactec.RTM. NR-660 and NR-730 manufactured by Becton Dickinson Diagnostic Instrument Systems, 383 Hillen Road, Towson, Md., are used in clinical microbiology laboratories to detect increases in carbon dioxide levels caused by microorganisms metabolizing a carbon source in a culture vial which holds the sample and medium. One primary use of the instrument is the detection of suspected septicemia caused by the presence of microorganisms in the blood of a patient. The presence of living blood cells in the sample being tested complicates these measurements by evolving additional carbon dioxide into the head space of a container for the sample. These clinical instruments do not distinguish between the carbon dioxide produced by the metabolism of a microorganism and the carbon dioxide produced by blood cell metabolism.
Automated instruments for the testing of blood cultures are described in U.S. Pat. Nos. 3,676,679 and 3,935,073 to Waters. The process involves the use of a growth medium containing a C.sup.14 carbon source which can be metabolized to produce gaseous C.sup.14 O.sub.2. A sample of blood to be tested is added to the growth medium and after a suitable period of incubation a portion of the gaseous atmospheres is tested for the presence of radioactive C.sup.14. The Waters patents do not address the reduction of background carbon dioxide formed by blood cell metabolism.
An improved process to those described in the above Waters patents is set forth in U.S. Pat. No. 3,854,041 to Waters, et al. The improvement requires the addition to the growth medium of a material such as sucrose, raffinose or glycylglycine in order to reduce the incidence of high background readings due to sterile blood.
U.S. Pat. No. 3,858,045 to Waters described the use of physical, osmotic or chemical lysis of blood cells to reduce the background CO.sub.2 readings due to blood metabolism. The '045 patent discloses non-ionic detergents such as polyoxyethylene, sorbitan and saponin as effective chemical lytic agents for use in radiometric culture media vials which effectively reduce the background carbon dioxide reading due to blood cell metabolism.
All of the above-described Waters and Waters et al. patents are directed towards the use and analyses of cultures in automated Bactec.RTM. radiometric C.sup.14 O.sub.2 detection instruments such as the Bactec.RTM. 460.
The method and apparatus utilized in the Bactec.RTM. NR-660 and NR-730, do not require the utilization of radioactive C.sup.14 in the culture medium. Rather, the growth of microorganisms in the cultures is detected by sampling the headspace gas present over the growth medium and measuring the carbon dioxide concentration in that gas by infrared analysis. This method and apparatus is described in U.S. patent application Ser. No. 597,633 (filed on Apr. 6, 1984). A modification of the method and apparatus described in Ser. No. 597,633 in which a direct non-invasive method for the analysis of the gaseous atmosphere container over the growth medium uses infrared absorption through the side walls of the container for the media is described in pending U.S. patent application Ser. No. 686,327 (filed Dec. 24, 1984). The disclosure of both of these patent applications are incorporated by reference in this application.
The inventors, herein, have successfully used the lytic agents described by U.S. Pat. No. 3,858,045 to Waters in the non-radiometric Bactec.RTM. NR-660 and NR-730 instruments. Saponin, a non-ionic detergent plant extract, was especially effective in reducing blood carbon dioxide backgrounds while remaining non-toxic to most organisms. It was found, however, that the lytic activity of saponin is decreased significantly by heat sterilization, losing about 1/2 of its lytic activity when saponin is present in dilute concentrations in media, i.e. less than 2 mg/ml.
The inventors, herein, have also discovered that the presence of cholesterol in blood serum significantly reduced the lytic activity of saponin. This discovery is consistent with the reported findings of Gogelein, et al., in Biochimica et Biophysica Acta, 773, 32-38 (1984), that saponin binds preferentially to cholesterol.
Accordingly, it is in object of this invention to provide a method for reducing the background carbon dioxide level in blood cultures due to the presence of mammalian cells, without inhibiting the metabolic activity of microorganisms present in the culture.
Another object of this invention is to provide a culture medium for reducing the background carbon dioxide level in blood cultures due to the presence of mammalian cells, without inhibiting the metabolic activity of microorganisms utilizing a non-ionic detergent which does not lose its lytic activity after the medium has been autoclaved.
A further object of this invention is to provide a culture medium for reducing the background carbon dioxide level in blood cultures due to the presence of mammalian cells without inhibiting the metabolic activity of microorganisms present in the culture, utilizing a non-ionic detergent which will not lose its lytic activity due to high cholesterol levels in the blood being sampled.